Radikal Therapeutics (RTX) is developing a novel bifunctional agent (R-503) that blocks two principal pathophysiolgoical pathways that contribute to traumatic brain injury (TBI). R-503 is both 1) a potent inhibitor of the nuclear DNA repair enzyme poly(ADP-ribose) polymeras (PARP) (IC50=20 nM), and 2) a dihydrolipoate (DHL)-based redox catalyst that is a superoxide dismutase mimetic, a catalase mimetic, and a peroxynitrite decomposition catalyst. The covalent linkage of both of the above functional moieties to form a single therapeutic agent is expected to create simultaneous and co-localized interruption of both the oxidant and PARP pathways of injury in TBI. The relevance of both pathways to the pathogenesis of TBI has been well established in experimental models of TBI and in the clinical setting. Overactivation of PARP, now confirmed in patients with TBI, consumes its substrate (NAD+), thereby depleting ATP stores and provoking energetic failure, loss of cellular homeostasis, neuronal necrosis, and brain infarction. The relevance of PARP activation to TBI is not merely in the initiation of brain injury, but also figures prominently in the recovery phase: Recent data indicate that PARP activation blocks the restorative response to TBI (neurogenesis) via its stimulation of microglial cells and its upregulation of NF-:B mediated transcription, which plays a central role in the expression of inflammatory cytokines, chemokines, adhesion molecules and inflammatory mediators, including matrix metalloproteases. Despite the apparent centrality of PARP activation to the initiation and recovery phases of TBI, there are clearly additional PARP-independent downstream effectors of redox-mediated injury. Accordingly, the level of clinical benefit afforded by stand-alone PARP inhibition is not likely to be sufficiently robust. Accordingly, we hypothesize that more comprehensive recovery from TBI wil require both: 1) the elimination of upstream oxidative and nitrosative redox stress, and 2) inhibition of downstream PARP activity. We will seek to confirm this hypothesis in an experimental model of TBI in which rats are subjected to a well- defined cortical contusion trauma, by comparing treatment with R-503, DHL (a redox catalyst), INO-1001 (a monofunctional PARP inhibitor), a combination of DHL and INO-1001, and a sham injury group. A resuscitation paradigm will be employed, whereby therapeutic agents will be introduced 2 h after cortical contusion and continued for 2 weeks. Neurobehavioral monitoring will include assessment of motor tasks (beam balance, beam walking, Morris water maze) on day 14, and post-mortem analysis of brain tissue for morphologic and biochemical evidence of redox and inflammatory injury, as manifested by histology score, levels of lipid peroxidation, protein nitrosation, PARP activation, apoptosis, and concentrations of TNF-1, MIP- 11, nuclear NF-: . Confirmation that RTX's bifunctional approach confers unique therapeutic superiority over a monofunctional PARP inhibitor and redox catalyst, and their combination, would provide justification for the continued development of R-503 as a first-in-class agent for emergent resuscitation and recovery of TBI. PUBLIC HEALTH RELEVANCE: The induction of cellular injury responses contributes importantly to the ultimate neurological outcome after severe head trauma. At present, there are no approved therapeutic measures that arrest inflammation and cell death programs that are triggered after cortical contusion. We are developing a novel drug that targets the basic mechanisms of this condition and will test this agent in a clinically-relevant animal model.